Standard immunoassays have been the dominant method of performing routine serum protein analyses for the last 60 years because they are a conceptually simple method capable of measuring antigens down to the pg/mL level. However, development times for immunoassays are quite long. Furthermore, differences among protein isoforms often occur in small hidden regions of the total structure. Immunological contact areas, epitopes, are also very small, making it difficult to interrogate these variable domains with antibodies. Moreover, antibodies do not clearly discriminate between partial cleavage products, auto-immune complexes, or post-translationally modified proteins. Elucidation of amino acid sequences and identification of post-translational modifications are critical measurements in the determination of protein function and activity. Mass spectrometers are capable of measuring these features, but only when proceeded by robust sample preparation processes.
It is rarely the case that an analyst is presented with a sample in a suitable form for injection into an LC/MS system. Most often the sample must be manipulated in some fashion to concentrate the compound of interest, remove interferences, enzymatically digest the sample to generate surrogate peptides with better mass spectrometric properties and modify the sample matrix/buffering system to achieve compatibility with LC separation and mass spectrometric detection systems (see FIG. 1).
With immunochromatographic analyses, the structural selectivity of antibodies can be used to bind and purify antigens from biological extracts then chromatography used to resolve proteins that differ by only small changes in structure. When combined with mass spectrometry, these methods enable detection of isoform variations and post-translational modifications; identification of these features being key to an understanding of protein function and activity. In a given run, hundreds of peaks can be resolved thus enabling high degrees of multiplexing.
It is frequently the case that affinity selection is performed using antibodies immobilized in a plate, on a surface, on particles or on magnetic beads. In addition, other methods may be used to purify targets with varying degrees of specificity, such as DNA and RNA based affinity systems, peptides, metals, metal oxides, lectins, and lipid binding proteins, as a nonexclusive selection. Furthermore, depending on the resolution of the mass spectrometer it is possible to perform less specific, bulk fractionations based on bulk properties such as size, hydrophilicity, hydrophobicity, charge, diffusion, and ion mobility. The result is that either the desired analytes of interest or undesired impurities in the sample are retained on the stationary phase. (Supelco (1998), Guide to Solid Phase Extraction). In a typical workflow, analytes of interest are retained on the stationary phase while the portion that passes through the stationary phase (the flow through) is discarded. In this case, the flow through contains unwanted materials. Once fractionated as such, the analyte of interest is then eluted from the stationary phase. Alternatively, analytes of interest are isolated using a stationary phase with affinity for the unwanted materials in the sample. In this case, the flow through is collected then analyzed.
Concentrating the compound of interest and removing interferences is an integral part of the sample preparation process. However, it is often the case that proteins are reduced to more easily identifiable peptide fragments by cleavage with proteolytic enzymes, the most popular being trypsin (LC-MS Based Detection of Differential Protein Expression, Leepika Tuli and Habtom Ressom J Proteomics Bioinform. 2009 Oct. 2; 2: 416-438). This process is often limited by the inefficiencies of solution digestion. Fundamentally, complete digestion using solution based approached is kinetically unfavorable. The drop in substrate concentration as a digestion nears completion makes it very difficult to obtain complete conversion to product. The process is further complicated by autolysis, a phenomenon that deactivates trypsin and changes its specificity over the course of the reaction.
Typically enzyme produces product at an initial rate that is approximately linear for a short period after the start of the reaction. As the reaction proceeds and substrate is consumed, the rate continuously slows. In order to push reaction kinetics in favor of a complete digestion trypsin can be immobilized onto a solid support. A large category of immobilized enzymes are used in protein chemistry analysis. For example, immobilized trypsin is used in LC/MS routinely to determine protein substrate's constituents. Whether it's a hospital, clinical lab or a biotech firm and whether it is diagnosis, discovery or validation, there are many situations where a quick and accurate contents analysis is beneficial. Traditionally, however, before subjecting the samples to immobilized enzyme reaction, a tedious sample pretreatment process is required. For example, alkylation, reduction or another denaturing maneuver is applied to the samples, often hours or days before analysis can be performed (see FIG. 1). Additionally, separation steps must be followed by an offline addition of reagents. These steps delay time to results while making initial steps in the sample preparation process incompatible with later steps. Ideally, separation and digestion could occur in a single device.
Thus, there is a need to provide a fast, robust and easy to use single reactor that can achieve both the analyte purification and protein digestion at the same reaction vessel. This disclosure provides working examples of such reactor that produces ready to be analyzed peptides in mass spectrometry.